Knockdown Of JARID2 Research Articles

453-P: Role of JARID2 in Regulating Inflammatory Genes via Epigenetic Mechanisms in Monocyte/Macrophages in Type 2 Diabetes

The activation of monocytes and macrophages plays a central role in chronic inflammation associated with type 2 diabetes mellitus (T2D) and cardiovascular disease. However, the precise epigenetic mechanisms involved are unclear. From RNA-seq analysis we found dysregulated expression of several genes, including downregulation of JARID2 in human CD14+ monocytes obtained from T2D patients versus controls. JARID2 protein functions as a transcription repressor by facilitating the recruitment of polycomb repressor complex 2 and associated histone H3K27-trimethylation to target genes. RT-qPCR and RNA-seq analysis showed that high glucose +TNF-α (HT) , key pathological factors in diabetes, downregulated JARID2 in human monocytes along with genes involved in inflammation and cytokine related signaling networks. Western blotting also showed HT reduces JARID2 protein levels in human monocytes. Furthermore, JARID2 knockdown with siRNAs increased proliferation and enhanced LPS and HT induced inflammatory genes in human monocytes. ATAC-seq revealed that HT increased chromatin-accessibility at promoters of inflammatory genes like IL1B that were upregulated by JARID2 knockdown. Interestingly, microRNA-seq analysis revealed that CD14+ monocytes from T2D patients showed increased expression of several microRNAs with complementarity to the JARID2 3’-UTR, indicating cross-talk between epigenetic factors and microRNA-dependent mechanisms. Mouse orthologous Jarid2 was downregulated in macrophages from diabetic db/db mice and in macrophages from normal C57BL/6 mice treated with HT, similar to human JARID2. These results demonstrate that JARID2 plays a key role in chromatin remodeling and restraining inflammatory genes in monocytes, and its downregulation in T2D can augment inflammation. Further, understanding of epigenetic regulators like JARID2 can lead to novel therapies for chronic inflammatory diabetic complications. Disclosure M.A.Reddy: None. V.S.Tanwar: None. L.Zhang: None. L.L.Lanting: None. M.Wang: None. P.Pandey: None. Z.Chen: None. X.Wu: None. R.Natarajan: None. Funding National Institutes of Health (DK065073) to RN AR-DMRI Endowment Pilot grant to MAR

Tumor mutation burden and JARID2 gene alteration are associated with short disease-free survival in locally advanced triple-negative breast cancer.

BackgroundIn locally advanced triple-negative breast cancer (TNBC), patients who did not achieve pathologic complete response (non-pCR) after neoadjuvant chemotherapy develop rapid tumor metastasis. Tumor mutation burden (TMB) is a potential biomarker of cancer therapy, though whether it is applicable to TNBC is still unclear.MethodsA total of 14 non-pCR TNBC patients were enrolled, and tissue samples from radical operation were collected. Of these, 7 cases developed disease progression within 12 months after operation [short disease-free survival (short DFS)], while others showed longer DFS over 1 year (long DFS). Next generation sequencing (NGS) analysis targeting 422 cancer-related genes and in vitro studies were performed.ResultsA total of 72 mutations were detected within 14 patients, which ranged from 1 to 8 per patient with a median mutations number of 5. The median number of mutations in the short-DFS group was higher than that in the long-DFS group (6.0 vs. 4.3; P=0.094). Furthermore, 6 gene mutation types were detected, with missense mutations displayed in the majority (36/72, 50.0%). No correlation between mutation type and DFS was found. Among 422 cancer-related genes, alterations in 30 genes were detected. TP53 (12/14, 85.7%) was the most common mutation gene in the entire cohort. RB1 mutations significantly occurred in patients with high Ki-67 scores (P=0.013). Additionally, 4 mutations of PTPN13 (57.1%, 4/7) and 3 of JARID2 (42.9%, 3/7) were only detected in the short-DFS group, while patients with JARID2 mutation had a significantly shorter DFS period (P=0.026). Experiments in vitro confirmed that JARID2 gene was widely expressed in various breast cancer cell lines. Knockdown of JARID2 in MD-MBA-231 cells by small interfering RNA (siRNA) decreased the expression of E-cadherin, and increased the levels of vimentin, MMP7, and MMP9.ConclusionsIn non-pCR TNBC, JARID2 mutation and TMB elevated in patients with short-DFS, indicating the potential prognostic biomarkers and therapeutic molecular targets for locally advanced TNBC.

Function of JARID2 in bovines during early embryonic development.

Histone lysine modifications are important epigenetic modifications in early embryonic development. JARID2, which is a member of the jumonji demethylase protein family, is a regulator of early embryonic development and can regulate mouse development and embryonic stem cell (ESC) differentiation by modifying histone lysines. JARID2 can affect early embryonic development by regulating the methylation level of H3K27me3, which is closely related to normal early embryonic development. To investigate the expression pattern of JARID2 and the effect of JARID2-induced H3K27 methylation in bovine oocytes and early embryonic stages, JARID2 mRNA expression and localization were detected in bovine oocytes and early embryos via qRT-PCR and immunofluorescence in the present study. The results showed that JARID2 is highly expressed in the germinal vesicle (GV), MII, 2-cell, 4-cell, 8-cell, 16-cell and blastocyst stages, but the relative expression level of JARID2 in bovine GV oocytes is significantly lower than that at other oocyte/embryonic stages (p < 0.05), and JARID2 is expressed primarily in the nucleus. We next detected the mRNA expression levels of embryonic development-related genes (OCT4, SOX2 and c-myc) after JARID2 knockdown through JARID2-2830-siRNA microinjection to investigate the molecularpathwayunderlying the regulation of H3K27me3 by JARID2 during early embryonic development. The results showed that the relative expression levels of these genes in 2-cell embryos weresignificantly higher than those in the blastocyst stage, and expression levels were significantly increased after JARID2 knockdown. In summary, the present study identified the expression pattern of JARID2 in bovine oocytes and at each early embryonic stage, and the results suggest that JARID2 plays a key role in early embryonic development by regulating the expression of OCT4, SOX2 and c-myc via modification of H3K27me3 expression. This work provides new data for improvements in the efficiency of in vitro embryo culture as well as a theoretical basis for further studying the regulatory mechanisms involved in early embryonic development.

Knockdown of JARID2 inhibits the viability and migration of placenta trophoblast cells in preeclampsia.

Protein Jumonji (JARID2) is a member of the Jumonji family of proteins and has been demonstrated to regulate cell proliferation and invasion. However, little is known about the role of JARID2 in the metastasis of placenta trophoblast cells. In the present study, the effect and the underlying molecular mechanism of JARID2 on trophoblast cell viability and invasion was investigated. The expression of JARID2 in placental tissues was analyzed by reverse transcription‑quantitative polymerase chain reaction and western blotting. HTR8/SVneo cells were transfected with si‑JARID2 or scramble for 24h. Cell viability, migration and invasion in HTR8/SVneo cells were then evaluated. The expression levels of matrix metallopeptidase 2 (MMP2), MMP9, phosphorylated phosphatidylinositol 3‑kinase (p‑PI3K), PI3K, phosphorylated AKT serine/threonine kinase 1 (p‑Akt) and Akt in HTR8/SVneo cells were also detected using western blotting. The results of the present study demonstrated that JARID2 is underexpressed in human preeclamptic placentas. The knockdown of JARID2 significantly inhibited the viability, migration and invasion of HTR8/SVneo cells. In addition, the knockdown of JARID2 significantly decreased the levels of phosphorylated PI3K and Akt in HTR8/SVneo cells. The results of the present study demonstrated that JARID2 may serve a role in the progression of preeclampsia. The knockdown of JARID2 inhibited the viability and invasion of trophoblast cells in preeclampsia by suppressing the PI3K/Akt signaling pathway. Therefore, JARID2 may serve as a novel potential target for treating preeclampsia.

Knockdown of JARID2 inhibits the proliferation and invasion of ovarian cancer through the PI3K/Akt signaling pathway.

Protein Jumonji (JARID2), a member of the family of JmjC domain-containing proteins, has been reported to serve an important role in tumor growth and metastasis. However, the expression pattern and role of JARID2 in ovarian cancer remains unclear. Therefore, in the present study, the role of JARID2 in ovarian cancer was investigated, as well as the underlying mechanisms. The results of the present study demonstrated that the expression of JARID2 is upregulated in human ovarian cancer cell lines. Furthermore, downregulation of JARID2 significantly suppressed proliferation, migration, invasion and epithelial‑mesenchymal transition in human ovarian cancer cells. Mechanistically, downregulation of JARID2 decreased the protein expression levels of phosphorylated phosphoinositide 3‑kinase (PI3K) and protein kinase B (Akt) in ovarian cancer cells. In conclusion the observations suggested that knockdown of JARID2 inhibited proliferation, migration and invasion invitro through the inactivation of the PI3K/Akt signaling pathway. Therefore, JARID2 may represent a potential therapeutic target for the treatment of ovarian cancer.

Jarid2 is essential for the maintenance of tumor initiating cells in bladder cancer.

Bladder cancer is the most common urologic malignancy in China, with an increase of the incidence and mortality rates over past decades. Recent studies suggest that bladder tumors are maintained by a rare fraction of cells with stem cell proprieties. Targeting these bladder tumor initiating cell (TICs) population can overcome the drug-resistance of bladder cancer. However, the molecular and genetic mechanisms regulating TICs in bladder cancer remain poorly defined. Jarid2 is implicated in signaling pathways regulating cancer cell epithelial-mesenchymal transition, and stem cell maintenance. The goal of our study was to examine whether Jarid2 plays a role in the regulation of TICs in bladder cancer. We found that knockdown of Jarid2 was able to inhibit the invasive ability and sphere-forming capacity in bladder cancer cells. Moreover, knockdown of Jarid2 reduced the proportion of TICs and impaired the tumorigenicity of bladder cancer TICs in vivo. Conversely, ectopic overexpression of Jarid2 promoted the invasive ability and sphere-forming capacity in bladder cancer cells. Mechanistically, reduced Jarid2 expression led to the upregulation of p16 and H3K27me3 level at p16 promoter region. Collectively, we provided evidence that Jarid2 via modulation of p16 is a putative novel therapeutic target for treating malignant bladder cancer.

Polycomb recruitment at the Class II transactivator gene.

The Class II Transactivator (CIITA) is the master regulator of Major Histocompatibility Class II (MHC II) genes. Transcription of CIITA through the IFN-γ inducible CIITA promoter IV (CIITA pIV) during activation is characterized by a decrease in trimethylation of histone H3 lysine 27 (H3K27me3), catalyzed by the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2). While EZH2 is the known catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is present at the inactive CIITA pIV, the mechanism of PRC2 recruitment to mammalian promoters remains unknown. Here we identify two DNA-binding proteins, which interact with and regulate PRC2 recruitment to CIITA pIV. We demonstrate Yin Yang 1 (YY1) and Jumonji domain containing protein 2 (JARID2) are binding partners along with EZH2 in mammalian cells. Upon IFN-γ stimulation, YY1 dissociates from CIITA pIV while JARID2 binding to CIITA pIV increases, suggesting novel roles for these proteins in regulating expression of CIITA pIV. Knockdown of YY1 and JARID2 yields decreased binding of EZH2 and H3K27me3 at CIITA pIV, suggesting important roles for YY1 and JARID2 at CIITA pIV. JARID2 knockdown also results in significantly elevated levels of CIITA mRNA upon IFN-γ stimulation. This study is the first to identify novel roles of YY1 and JARID2 in the epigenetic regulation of the CIITA pIV by recruitment of PRC2. Our observations indicate the importance of JARID2 in CIITA pIV silencing, and also provide a novel YY1-JARID2-PRC2 regulatory complex as a possible explanation of differential PRC2 recruitment at inducible versus permanently silenced genes.

MiRNA-204 drives cardiomyocyte proliferation via targeting Jarid2

ObjectivesIn mammals, the heart grows by hypertrophy but not proliferation of cardiomyocytes after birth. The paucity of cardiomyocyte proliferation limits cardiac regeneration in a variety of heart diseases. To explore the efficient strategies that drive cardiomyocyte proliferation, we employed in vitro and in vivo models to investigate the function of miRNA-204, which was demonstrated to regulate the proliferation and differentiation of human cardiac progenitor cells in our previous study. Methods and resultsmiRNA-204 overexpression markedly promoted cardiomyocyte proliferation in both neonatal and adult rat cardiomyocytes in vitro. Transgenic mice with the cardiac-specific overexpression of miRNA-204 exhibited excessive cardiomyocyte proliferation throughout the embryonic and adult stages, leading to a pronounced increase in ventricular mass. Accordingly, the cell cycle regulators, including Cyclin A, Cyclin B, Cyclin D2, Cyclin E, CDC2 and PCNA, were upregulated in miRNA-204 transgenic embryonic hearts. Furthermore, we demonstrated that miRNA-204 directly targeted Jarid2. Knockdown of Jarid2 mimicked the pro-proliferative effect of miRNA-204 overexpression on cultured rat cardiomyocytes, whereas enhanced expression of Jarid2 conferred the myocytes with substantial resistance to proliferation by miRNA-204 overexpression. ConclusionOur findings identify a conserved role for miRNA-204 in regulating cardiomyocyte proliferation by targeting the Jarid2 signaling pathway.

JARID2 inhibits leukemia cell proliferation by regulating CCND1 expression.

It has recently been shown that JARID2 contributes to the malignant character of solid tumors, such as epithelial-mesenchymal transition in lung and colon cancer cell lines, but its role in leukemia progression is unexplored. In this study, we explored the effect and underlying molecular mechanism of JARID2 on leukemia cell proliferation. Real-time PCR and Western assay were carried out to detect JARID2 and CCND1 expression. Cell number and cell cycle change were detected using hemocytometer and flow cytometry, and a ChIP assay was utilized to investigate JARID2 and H3K27me3 enrichment on the CCND1 promoter. JARID2 is down-regulated in B-chronic lymphocytic leukemia (B-CLL) and acute monocytic leukemia (AMOL), and knockdown of JARID2 promotes leukemia cell proliferation via acceleration of the G1/S transition. Conversely, ectopic expression of JARID2 inhibits these malignant phenotypes. Mechanistic studies show that JARID2 negatively regulates CCND1 expression by increasing H3K27 trimethylation on the CCND1 promoter. Our findings indicate that JARID2 is a negative regulator of leukemia cell proliferation, and functions as potential tumor suppressor in leukemia.

JARID2 is involved in transforming growth factor-beta-induced epithelial-mesenchymal transition of lung and colon cancer cell lines.

Histone methylation plays a crucial role in various biological and pathological processes including cancer development. In this study, we discovered that JARID2, an interacting component of Polycomb repressive complex-2 (PRC2) that catalyzes methylation of lysine 27 of histone H3 (H3K27), was involved in Transforming Growth Factor-beta (TGF-ß)-induced epithelial-mesenchymal transition (EMT) of A549 lung cancer cell line and HT29 colon cancer cell line. The expression of JARID2 was increased during TGF-ß-induced EMT of these cell lines and knockdown of JARID2 inhibited TGF-ß-induced morphological conversion of the cells associated with EMT. JARID2 knockdown itself had no effect in the expression of EMT-related genes but antagonized TGF-ß-dependent expression changes of EMT-related genes such as CDH1, ZEB family and microRNA-200 family. Chromatin immunoprecipitation assays showed that JARID2 was implicated in TGF-ß-induced transcriptional repression of CDH1 and microRNA-200 family genes through the regulation of histone H3 methylation and EZH2 occupancies on their regulatory regions. Our study demonstrated a novel role of JARID2 protein, which may control PRC2 recruitment and histone methylation during TGF-ß-induced EMT of lung and colon cancer cell lines.

Abstract 675: Jarid2 is a novel target for lung cancer therapy.

Abstract Increasing evidence indicates that aberrant expression/activity of polycomb repressive-complex-2 (PRC-2) contributes to the pathogenesis of a variety of human malignancies including lung cancers. In recent studies we have demonstrated that depletion of EZH2- a core component of PRC-2, either by biochemical or pharmacologic techniques inhibits the malignant phenotype of lung cancer cells, indicating that PRC-2 is a target for epigenetic therapy of lung cancer. In the present study, we sought to further examine the potential efficacy of targeting PRC-2 in lung cancers via depletion of Jarid2, which modulates gene expression in stem cells in part via targeting PRC-2 to DNA. Quantitative RT-PCR and immunoblot experiments demonstrated over-expression of Jarid2 in cultured lung cancer cells and primary lung cancers relative to normal respiratory epithelia or histologically normal lung tissues. In subsequent experiments, shRNA techniques were used to inhibit Jarid2 expression in lung cancer cells. Quantitative RT-PCR and immunoblot experiments demonstrated that knock-down of Jarid2 had relatively modest effects on global H3K27Me3 levels in lung cancer cells. These finding were in contrast to knock-down of EZH2, which markedly inhibited H3K27 trimethylation in these cancer cells. Interestingly, knock-down of Jarid2 inhibited in vitro proliferation of lung cancer cells by approximately 70% relative to respective controls, and significantly diminished growth of subcutaneous lung cancer xenografts in athymic nude mice. The anti-proliferative effects of Jarid2 knock-down were relatively comparable to those observed following depletion of EZH2 in lung cancer cells. Jarid2 knock- down only modestly enhanced cytotoxicity mediated by DZNep in lung cancer cells. Microarray experiments are currently in progress to compare gene expression profiles in lung cancer cells mediated by Jarid2 knock-down relative to EZH2 depletion or DZNep treatment, and to delineate the mechanisms by which Jarid2 enhances the malignant phenotype of lung cancer cells. Collectively, these preliminary findings warrant further analysis of Jarid2 expression during pulmonary carcinogenesis, and suggest that Jarid2 may be a novel target for lung cancer therapy. Citation Format: Young K. Hong, Mary Zhang, Scott Atay, Manish Raiji, Trevor Upham, Julie A. Hong, Mahadev Rao, David S. Schrump. Jarid2 is a novel target for lung cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 675. doi:10.1158/1538-7445.AM2013-675

PRC2 Complexes with JARID2, MTF2, and esPRC2p48 in ES Cells to Modulate ES Cell Pluripotency and Somatic Cell Reprograming

Polycomb repressive complex two (PRC2) has been implicated in embryonic stem (ES) cell pluripotency; however, the mechanistic roles of this complex are unclear. It was assumed that ES cells contain PRC2 with the same subunit composition as that identified in HeLa cells and Drosophila embryos. Here, we report that PRC2 in mouse ES cells contains at least three additional subunits: JARID2, MTF2, and a novel protein denoted esPRC2p48. JARID2, MTF2, and esPRC2p48 are highly expressed in mouse ES cells compared to differentiated cells. Importantly, knockdowns of JARID2, MTF2, or esPRC2p48 alter the level of PRC2-mediated H3K27 methylation and result in the expression of differentiation-associated genes in ES cells. Interestingly, expression of JARID2, MTF2, and esPRC2p48 together, but not individually, enhances Oct4/Sox2/Klf4-mediated reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells, whereas knockdown or knockout of JARID2, MTF2, or esPRC2p48 significantly inhibits reprogramming. JARID2, MTF2, and esPRC2p48 modulate H3K27 methylation and facilitate repression of lineage-associated gene expression when transduced into MEFs, and synergistically stimulate the histone methyltransferase activity of PRC2 in vitro. Therefore, these studies identify JARID2, MTF2, and esPRC2p48 as important regulatory subunits of PRC2 in ES cells and reveal critical functions of these subunits in modulating PRC2's activity and gene expression both in ES cells and during somatic cell reprogramming.

Jarid2 and PRC2, partners in regulating gene expression

The Polycomb group proteins foster gene repression profiles required for proper development and unimpaired adulthood, and comprise the components of the Polycomb-Repressive Complex 2 (PRC2) including the histone H3 Lys 27 (H3K27) methyltransferase Ezh2. How mammalian PRC2 accesses chromatin is unclear. We found that Jarid2 associates with PRC2 and stimulates its enzymatic activity in vitro. Jarid2 contains a Jumonji C domain, but is devoid of detectable histone demethylase activity. Instead, its artificial recruitment to a promoter in vivo resulted in corecruitment of PRC2 with resultant increased levels of di- and trimethylation of H3K27 (H3K27me2/3). Jarid2 colocalizes with Ezh2 and MTF2, a homolog of Drosophila Pcl, at endogenous genes in embryonic stem (ES) cells. Jarid2 can bind DNA and its recruitment in ES cells is interdependent with that of PRC2, as Jarid2 knockdown reduced PRC2 at its target promoters, and ES cells devoid of the PRC2 component EED are deficient in Jarid2 promoter access. In addition to the well-documented defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing.

Jarid2/Jumonji Coordinates Control of PRC2 Enzymatic Activity and Target Gene Occupancy in Pluripotent Cells

Polycomb Repressive Complex 2 (PRC2) regulates key developmental genes in embryonic stem (ES) cells and during development. Here we show that Jarid2/Jumonji, a protein enriched in pluripotent cells and a founding member of the Jumonji C (JmjC) domain protein family, is a PRC2 subunit in ES cells. Genome-wide ChIP-seq analyses of Jarid2, Ezh2, and Suz12 binding reveal that Jarid2 and PRC2 occupy the same genomic regions. We further show that Jarid2 promotes PRC2 recruitment to the target genes while inhibiting PRC2 histone methyltransferase activity, suggesting that it acts as a "molecular rheostat" that finely calibrates PRC2 functions at developmental genes. Using Xenopus laevis as a model we demonstrate that Jarid2 knockdown impairs the induction of gastrulation genes in blastula embryos and results in failure of differentiation. Our findings illuminate a mechanism of histone methylation regulation in pluripotent cells and during early cell-fate transitions.